DOI: https://doi.org/10.21203/rs.3.rs-2151004/v1
Background: This study investigates the effects of soybean meal fermented by Enterococcus faecium as a replacement for soybean meal on growth performance, apparent total tract digestibility, blood profile and gut microbiota of weaned pigs.
Methods: Eighty piglets (weaned at 21 days) [(Landrace x Yorkshire) x Duroc] with average body weight of 6.52 ± 0.59 kg) were selected and assigned to 4 treatments / 4 replicate pens (3 barrows and 2 gilts). The four diets (SBM, 3%, 6% and 9% FSBM) were formulated using FSBM to replace 0%, 3%, 6% and 9% of SBM, respectively. The trial lasted for 42 days (phase 1 (days 0-7), phase 2 (8-21), and phase 3 (days 22-42).
Results: Result showed that supplemental FSBM increased (P < 0.05) the body weight gain (BWG) for piglets at day 7, 21 and 42 and ADG at days 1-7, 8-21, 22- 42 and 1-42, and ADFI at days 8-21,22-42 and 1-42 and G: F at days 1-7, 8-21 and 1-42, and crude protein, dry matter, and energy digestibility at day 42, and the lowered (P< 0.05) diarrhea at days 1-21 and 22-42. The concentration of glucose levels, WBC, RBC and lymphocytes were increased while, concentration of BUN level in the serum was lowered in the FSBM treatment compared to the SBM group (P <0.05). Microbiota sequencing found that FSBM supplementation increased the microbial Shannon, Simpsons and Chao indexs, (P< 0.05) and the abundances of the phylam Firmicutes, and genera prevotella, Lactobacillus, Lachnospiraceae and Lachnoclostridium(P< 0.05), lower in the abundances of the phylam bacteroidetes, Proteobacteria, genera Escherichia-Shigella, Clostridium sensu stricto1, Bacteroides and Parabacteroides (P <0.05).
Conclusions: Overall, FSBM replacing SBM improved the growth performance, nutrient digestibility, and blood profiles; perhaps via altering the faecal microbiota and its metabolites in weaned pigs. The present study provides theoretical support for applying FSBM at 6 to 9% promote immune characteristics and regulate intestinal health in weaning piglets.
In the swine husbandry, weaning produce gut system dysfunctions and results from inconstant impairment of the gut barrier function, oxidative stress and absorption as a result of poor growth, diarrhoea and other diseases [1, 2]. Weaning pigs are immediately required to undergo a change from high digestible sow’s milk to solid diets as well as complex protein [3, 4]. Therefore, it is important to explore potential protein sources that should be added to diets to alleviate the weaning stress of piglets. The high cost, finite supply, and unstable variation of sources animal protein had become major reasons for limiting its supplement in diets on weaning pigs. Soybean meal (SBM) is an important protein feed ingredient in livestock diet SBM, but variety of antinutritional factors (ANF) as well as β-conglycinin, glycinin and trypsin inhibitor, which would result to immune responses, digestive disorders and negative effects on animal health [3, 5]. Cheng et al. [6] and Li et al. [7] reported that bacterial fermentation could reduce content by ANFs and improving on nutritional quality and nutrient bioavailability. Fermented soybean meal (FSBM), a manufactured product mixed with solid SBM, liquid phases and the vaccinating the mixture with E. faecium [8] could improve protein quality and reduce on ANFs levels of SBM with solid state fermentation [9]. It has been reported that FSBM has partial or total replacement of SBM to improve the growth performance and apparent tract total digestibility (ATTD) of crude protein and gross energy immune and antioxidant capacity in weaning pigs [2]. Jeong et al. [10] found that compared with SBM, FSBM had improve the growth performance and the apparent ileal digestibility of amino acids and other nutrients in weaning piglets. Moreover, compared with SBM, FSBM showed greater concentration of CP and amino acid and reduce trypsin inhibitor and ANFs [11].
The ideal situation would be to select feed probiotics that would improve soybean meal quality and enhance resistance to weaning stress by bolstering the gut microbiota. Using Enterococcus faecium (E. faecium) to produce a fermented soybean meal (FSBM) in weaning pigs has not been stated earlier and a probiotic-fermented SBM might show some different effects on piglets. In the current study was to compare the effects of dietary SBM and FSBM on growth performance, ATTD, blood profile and gut microbiota of weaning pigs were explored.
Experimental protocols were approved by Dankook University Animal Care Committee (Permit number DK-2-1936). The SBM and FSBM were sourced by Feedup Com, Ltd, Republic of Korea. The analyzed nutrient composition of SBM and FSBM is presented in Table S1.
Eighty [(Landrace x Yorkshire) x Duroc] crossed weaned piglets (21 of age; 6.52 ± 0.59 kg) were randomly selected and allocated into 4 diets with 4 pens replicates according to the average initial body weight and sex, and each pen has 5 piglets (3 barrows and 2 gilts). Four diets (SBM, 3% FSBM, 6% FSBM and 9% FSBM) were formulated using FSBM to replace 0%, 3%, 6% and 9% of SBM, respectively. The trial lasted for 42 days (phase 1 (days 0–7), phase 2 (8–21) and phase 3 (days 22–42). Basal diets were formulated to meet the NRC (2012) requirements (Table S2, S3 and S4). Throughout the trial, all pigs had free access to feed and water, and the room temperature was maintained at 24 to 26°C with 60–70% humidity, respectively. Each piglets were weighed on days 0, 7, 21 and 42 and feed consumption was also recorded on a pen basis to determine average daily gain (ADG), average daily feed intake (ADFI) and Gain to feed ratio (G:F = ADFI/ADG). The piglets’ anuses were checked one by one at 09:00and 17:00 daily during the experiment to observe and recorded any fecal contamination and redness. The number of piglets with diarrhea per treatment was counted at the end of the experiment and the diarrhea rate was calculated with the following formulation:
Diarrhea rate (%) = 100% × total number of piglets with diarrhea/ (total number of piglets × number of days).
To determine dry matter (DM), crude protein (CP), and energy digestibility, chromium oxide was added to the diet as an indigestible marker at 2 g/kg of the diet for7 d prior to fecal collection. Fecal samples were collected from 8 pigs randomly selected per treatment via rectal massage, and the sample was stored in a freezer at − 20°C and were dried in a 65°C for 72 h and the feed and fecal samples were grounded to passed through 1-mm sieve for the measurement of dry matter (DM), CP, and energy of FSBM, SBM, diets and feces samples were determined following to the Association of Official Analytical Chemists [12] procedures.
At 06:00 on days 21 and 42, two pigs for each replicate was randomly selected to collect blood from the jugular vein and subsequently centrifuged at 3,000 × g for 15 min at 4°C to obtain the serum sample and was kept at − 80°C until analysis. The concentrations of lymphocyte counts, Red Blood cells (RBC), White blood cell (WBC) BUN, and glucose in serum were analyzed as the operation steps of the kit (ADVIA 120; Bayer, Tarrytown, NY, USA). And the serum creatinine concentration was analyzed by automatic biochemical analyzer (Beckman Instruments, Inc., Brea, CA, US).
On day 42, each group selected 6 pigs of fresh fecal samples (n = 6) for microbiota analysis. Genomic DNA of fecal samples was extracted by using a DNA Kit (Omega Bio-tek, Norcross, GA, USA), according to manufacturer’s instructions. The quantity and quality of extracted genomic DNA were checked using a UV spectrophotometer (Mecasys, Daejeon, Korea). Amplification and sequencing of the V5–V6 hypervariable region of the 16S rRNA gene was performed using an Illumina MiSeq platform (Illumina, San Diego, CA, USA). We performed alpha-diversity and taxonomic analyses of the raw paired-end sequences using EZBioCloud pipeline (13). Then the samples were grouped into microbiome taxonomic profile sets for further analyses. Relative abundance cut-offs at the phylum and genus levels were set to 0.1%. Charts depicting the results from the alpha-diversity and taxonomic analyses were generated using GraphPad Prism software version 9.0 (GraphPad, San Diego, California, USA).
All results are presented as mean ± standard deviation (SD). GraphPad 9.0 was used figures. Statistical analyses were performed using IBM SPSS 20.0 (SPSS Inc., Chicago, IL, USA) and the differences between treatments were compared with one-way ANOVA followed by Dunnett’s multiple comparison procedure. For all tests, P < 0.05 was considered as significant, while 0.05 > P < 0.10 as a tendency.
As shown the Fig. 1, the BW of piglets fed dietary FSBM were also higher than SBM. The 3% and 6% FSBM (P < 0.05) during days 7, 21 and 42, 9% FSBM (P < 0.05) during days 7 and 22, (P < 0.01) during day 42 all showed significant increases. Compared with SBM treatments, piglets fed 3, 6 and 9% FSBM (P < 0.05) on the higher ADG, ADFI and G:F during days 1–7, 8–21, 22–42 and 1–42. Importantly, the diarrhea rate lower in FSBM treatments (P < 0.05).
Dietary supplementation with 3, 6 and 9% FSBM increased (P < 0.05) the ATTD of CP, DM and energy compared to the SBM (Fig. 2). Higher (P < 0.05) DM was found in the piglets dietary supplemented with 6% FSBM than 3 and 9% FSBM. However, 9% FSBM diet showed elevated CP levels than piglets fed 3 and 6% FSBM.
As illustrated in Fig. 3, piglets diets FSBM diets had increased (P < 0.05) the glucose levels, WBC, RBC and lymphocytes and lowered (P < 0.05) the BUN level on days day 21 and 42. High (P < 0.05) glucose, RBC, WBC, Lymphochte and lower BUN compared with SBM was observed (Fig. 3) in serum gathered from days 21 and 42 piglets supplemented with the 6% FSBM. The Lymphocyte of serum in day 42 piglets fed diets with 9% FSBM was higher than in 3 and 6% FSBM. BUN was lower in the latter two group than in 3 and 6% FSBM. The Creatinine was not influenced in the serum by feeding 3, 6 and 9%FSBM.
The OTUs Venn analysis identified 21, 28, 46 and 69 unique OTUs among FSBM and SBM treatments, respectively (Fig. 4A). Figure 4B illustrates the results of alpha diversity analysis of which the Shannon, Simpsons and Chao indices were improved (P < 0.05) on FSBM compared to the SBM.
As shown in Fig. 5, the phylum levels analysis illustrated that the dietary supplementation of FSBM higher in the abundance of Firmicutes (P < 0.05) and lower the abundance of Bacteroidetes and Proteobacteria (P < 0.05). At the genus level, FSBM group increased Lactobacillus, prevotella, Lachnospiraceae and Lachnoclostridium (P < 0.05) in fecal microbiota (P < 0.05). Compared with SBM treatment, FSBM treatments lower in the abundance of Escherichia-Shigella, Clostridium sensu stricto1, Bacteroides and Parabacteroides (P < 0.05) (Fig. 6).
Sources of plant protein like FSBM have been extensively used in the diets of weaning pigs to enhance growth and immune status [2, 4]. Earlier studies, illustrated that dietary FSBM supplementation increased the growth performance on weaning pigs [14, 15, 16] and broilers [6]. Zhu et al. [17] reported increased growth performance and reduced diarrhea in weaning piglets due to dietary FSBM supplementation. In current study, the BW, ADG, and ADFI were improved and the diarrhea decreased in weaning pigs supplemented with FSBM treatments. Pigs fed with FSBM could improve nutrient digestibility that may explain an enhanced ADG in weaning pigs.
The BUN concentration and producing are affected by way of protein catabolism, and its concentrating is negatively associated with digestibility of proteins and amino acids [18]. Creatinine exists a natural waste result arises from the muscles and is eliminated from the body through kidney. Pigs fed FSBM showed decreased diarrhea score and the glucose levels improved which was in accordance with earlier studies [19, 17]. Intriguingly, serum blood urea nitrogen reduced in FSBM treatments compared with the control. This shown that the fermenting procedure change nitrogen distribution inside the feed [20]. The levels of white blood cells, lymphocytes, were also increased, in the current studies. Lymphocyte growth exists as a major phase during the immune reaction in an animal and a proliferative reaction is a specific antigen [21]. Gizzarelli et al. [22] and Wang et al. [23] reported that weaning pig immunity is lower on β-conglycinin is not adequate deactivated during fermentation. Our results found a higher immune resistance on piglets this directly corresponded with decrease in glycinin and β-conglycinin on FSBM. Therefore, overall growth performance and blood profiles were in accordance with each other.
Alpha diversity is characterized as the diversity of species richness in a specific area or ecosystem [24, 25]. In our study, Simpson, Shannon, and Chao1 richness were significantly increased in FSBM groups compared with the SBM group, which can explain by the suitable growth ecosystem for the microbes because of the organic acid and probiotic of the FSBM group. Bacteroidetes, Firmicutes, Actinobacteria, Proteobacteria, and Tenericutes were the most pre-dominant bacteria phylum in the piglets [26]. In the current study, FSBM group shapes gut microbiota in weaning pigs, including lower in the abundances of the phylam Bacteroidetes, and Proteobacteria and the genera Escherichia-Shigella, Clostridium sensu stricto1, Bacteroides and Parabacteroides, and a higher in the abundances of the phylam Firmicutes and the genera Lactobacillus, prevotella, Lachnospiraceae and Lachnoclostridium. Bacteroidetes, Proteobacteria and Firmicutes as three major communities, are essential to growth performance and energy metabolism homeostasis [27, 28]. Shin et al. [29] found that a lower in the abundance of the phyla Proteobacteria in the gut of healthy humans. Therefore, Litvak et al. [30] reported that increase in Proteobacteria abundance have been associated with in humans with gut inflammation, colorectal cancer, irritable bowel syndrome and metabolic syndrome and could be bacterial signature of gut dysbiosis [29]. The abundance of the phyla Proteobacteria contains many possible opportunistic pathogens, as well as Campylobacter spp, Klebsiella spp, Escherichia, and Salmonella and its rise could be shown as a potential indicator of gut diseases. The Firmicutes abundance have been evidence to be positive relationship with energy and active transport, facilitated diffusion, endocytosis and passive diffusion, whereas improve in fecal Proteobacteria and Bacteroidetes is associated with inferior nutrient digestibility [2]. Therefore, higher abundances of the phyla Firmicutes along with lower abundances of the phyla Proteobacteria and Bacteroidetes might promote nutrient digestibility in weaning pigs. Eren et al. [31] reported that the herbivores have an increase abundance of Lachnospiraceae than carnivores in animal. Vacca et al. [32] reported that reduce abundance of Lachnospiraceae, while multiple sclerosis and ulcerative colitis patients. All participants of Lachnospiraceae are anaerobic, hemoorganotrophic and fermentative and could degrade non-starch polysaccharides and butyrate and acetic acid. Butyrate provide the major energy source for intestinal epithelial cell growth, increased intestinal protection mediated epithelial cells and favoring the suppression of inhibits inflammatory responses [33, 34]. Stanley et al. [35] showed that Lachnospiraceae is associated with improve growth performance in animals. The abundances of the genera Lachnoclostridium—butyric-acid-producing microbes that have been associated in the mitigation on intestinal inflammation- were better in the FSBM groups [7]. The Lactobacillus as a possibility probiotic, possess the opposition to pathogen, anti– inflammatory, antioxidant capacity, and capability to higher of fecal microbiota [36, 37]. Zhu et al. [26] reported an improved Lactobacilli and totality anaerobic bacteria counts in the gut microbiota weaning piglets due to dietary FSBM supplementation. In the current study, inclusion of FSBM treatments an increased in the abundances of the genera Lactobacillus were significantly increased glucose levels and decreased BUN. Lactobacillus is familiar to have a positive influence on the GIT, growth performance, and nutrient digestibility in pigs and regularly used as probiotics in livestock production [38, 25]. Yan et al. [39] reported that Lactobacillus could commonly increase growth performance and the GIT of animals by defense the intestine from pathogens and encourage efficient nutrient and energy extraction by the host. The genus Prevotella is saccharolytic and produce succinic and acetic acids as ending fermentation products (40). Prevotella specialized in degrading fiber diet, which had also been correspondent with could improve intestinal immune and decrease diarrhea [41]. Wu et al. [42] reported evidence indicated a close relationship among Prevotella and long period of time carbohydrates diets or carbohydrates from fiber-rich diets. Feng et al. [43] and Forsyth et al. [44] reported a higher Prevotella abundance, the higher the mucin composite content, but bacterial toxins would reduce gut penetrability and the sensitivity of the region-specific gut to systemic exposure. Accordingly, higher in the Prevotella abundances in FSBM it could be helpful in improve on growth performance in weaning piglets. The relative abundance of Clostridium sensu stricto1 and bacteoides showed a positively correlated with the frequency of diarrhea. Clostridium is the main cause of diarrhoea in humans and is responsible for community-acquired out breaks [45]. Harlow et al. [46] reported that Clostridium perfringens, Clostridium difficile and Salmonella spp. are the most common microbes associated with diarrhoea. In our study, lower in the abundances of the genera Clostridium sensu stricto1 in the FSBM group, and was positively correlated with decreased diarrhoea. Another result of FSBM groups were a decrease Escherichia-Shigella, and lower Escherichia-Shigella was main participant of the decreased in Proteobacteria abundance compared to the SBM treatments. The genera Escherichia–Shigella comprises an opportunistically pathogenic bacterium. Sousa et al. [47] and Gong et al. [48] reported a the genera Escherichia–Shigella could destroy the gut structure and must be pro-inflammatory actions through multiple pathways and like the secretion of virulence factors, consequent in the improved danger of infection and diarrhoea in the host. Parabacteroides and Bacteroides, which occur at the initial phase of the lifetime, have been informed to generate gamma amino butyric acid, closely related to growth [49]. A higher abundance of the genera Bacteroides is commonly used in the events of colorectal cancer, functional gastrointestinal disorders and ulcerative colitis [50]. Therefore, the biological results found that appropriate inclusion of FSBM in diets can inhibit the gut pathogens (such as Escherichia–Shigella, Bacteroides and Parabacteroides) and enhance beneficial bacteria (such as Prevotella and Lactobacillus) and further enhance the immunity and health status of weaning piglets.
In conclusion, FSBM replacing SBM improved the growth performance, apparent total tract digestibility, blood profiles, possibly via the altering gut microbiota profile and its metabolites in weaned piglets. The present study provides theoretical support for applying FSBM at 6 to 9% promote immune characteristics and regulate intestinal health in weaning piglets.
All data generated or analyzed during this study are available from the corresponding author on reasonable request.
Experimental protocol of this study was approved by the Dankook University Animal Care Committee (Permit number DK-2-1936).
DATA AVAILABILITY STATEMENT
All data generated or analyzed during this study are available from the corresponding author on reasonable request.
ETHICS STATEMENT
Experimental protocol of this study was approved by the Dankook University Animal Care Committee (Permit number DK-2-1936).
AUTHOR CONTRIBUTIONS
MM, JHP and IHK: Conceptualization and designed the trials. MM: writing – original draft preparation, performed the animal trials, MM, IHK, JHP and KH: Software, Methodology, Formal analysis, Writing – review and editing, IHK: Supervision. All authors contributed to the article and approved the submitted version.
Disclosure statement
None.